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Objectives: Monitoring of therapeutic antibody adalimumab (ADL) and of anti-adalimumab antibodies (AAA) in autoimmune diseases patients' sera has achieved increased attention since several studies showed a correlation between AAA levels and treatment failure. We evaluated a new surface plasmon resonance (SPR)-based method that, with slight changes in the analysis condition and in the ligand immobilized on the chip surface, allows to monitor both AAA and ADL. This new label-free method does not require sample pretreatments, and it is fully automated, only requiring the preparation of the chip, which can be used for multiple analysis, and the preparation of the sample sets., Design & Methods: Sera from ADL-treated patients (n = 47) and controls (n = 13) were included in this study. Quantitative analysis of AAA and ADL were performed separately using a new SPR-biosensor, and a commercially available ELISA kit. Agreement was defined by overall, positive, and negative agreement. Wilson Score was used to calculate confidence intervals (CI) on binomial probability and Spearman's rho and Bland-Altman test were used to assess correlations., Results: ELISA and SPR-based assay were able to identify circulating AAA in ADL-treated patients, with the percentage of positivity varying among the methods, with an overall agreement of 79%. AAA were detected in 18 (38 %) out of the 47 treated patients by the ELISA whereas SPR-based assay detected 10 (21 %) out of 47 samples., Conclusions: Real-time label free SPR-based protocol for both AAA and ADL quantification has been set-up. Although quantitative differences were observed when compared with ELISA, the agreement among methodologies was high, particularly for ADL quantification within the therapeutic window of the drug., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Multiple sclerosis (MS) is an inflammatory and autoimmune disorder, in which an antibody-mediated demyelination mechanism plays a critical role. We prepared two glucosylated peptides derived from the human myelin proteins, that is, oligodendrocyte-myelin glycoprotein (OMGp) and reticulon-4 receptor (RTN4R), selected by a bioinformatic approach for their conformational homology with CSF114(Glc), a designed β-turn antigenic probe derived from myelin oligodendrocyte glycoprotein (MOG), a glycoprotein present in the CNS. This synthetic antigen is specifically recognized by antibodies in sera of MS patients. We report herein the antigenic properties of these peptides, showing, on the one hand, that MS patient antibodies recognize the two glucosylated peptides and, on the other hand, that these antibodies cross-react with CSF114(Glc) and with the previously described hyperglucosylated nontypeable Haemophilus influenzae bacterial adhesin protein HMW1ct(Glc). These observations point to an immunological association between human and bacterial protein antigens, underpinning the hypothesis that molecular mimicry triggers the breakdown of self-tolerance in MS and suggesting that RTN4R and OMGp can be considered as autoantigens., (© 2023 European Peptide Society and John Wiley & Sons Ltd.)

The involvement of Myelin Basic Protein (MBP) in Multiple Sclerosis (MS) has been widely discussed in the literature. This intrinsically disordered protein has an interesting α-helix motif, which can be considered as a conformational epitope. In this work we investigate the importance of the helical structure in antibody recognition by MBP peptides of different lengths. Firstly, we synthesized the peptide MBP (81-106) (1) and observed that its elongation at both N- and C-termini, to obtain the peptide MBP (76-116) (2) improves IgM antibody recognition in SP-ELISA, but destabilizes the helical structure. Conversely, in competitive ELISA, MBP (81-106) (1) is recognized more efficiently by IgM antibodies than MBP (76-116) (2), possibly thanks to its more stable helical structure observed in CD and NMR conformational experiments. These results are discussed in terms of different performances of peptide antigens in the two ELISA formats tested., Competing Interests: Author HR is employed by Fischer Analytics GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Staśkiewicz, Quagliata, Real-Fernandez, Nuti, Lanzillo, Brescia-Morra, Rusche, Jewginski, Carotenuto, Brancaccio, Aharoni, Arnon, Rovero, Latajka and Papini.)

Tentacle-like polymers decorated with several copies of peptide antigens can be interesting tools for increasing the ability to capture circulating antibodies in patient sera, using cooperative effects for stronger avidity. We previously showed that antibodies from multiple sclerosis (MS) patient sera preferentially recognize hyperglucosylated adhesin protein HMW1ct of non-typeable Haemophilus influenzae (NTHi). We selected the C-terminal HMW1ct(1347-1354) minimal epitope and prepared the diglucosylated analogue Ac-KAN(Glc)VTLN(Glc)TTG-K(N 3 )-NH 2 to graft a 40 kDa dextran scaffold modified with glycidyl-propargyl moieties to perform a copper catalyzed alkyne-azide coupling reaction (CuAAC). Quantitative NMR measurements allowed the characterization of the peptide loading (19.5 %) on the multivalent dextran conjugate. This novel polymeric structure displayed optimal capturing properties of both IgG and, more interestingly, IgM antibodies in MS sera. Specific antibodies from a representative MS serum, were successfully depleted using a Sepharose resin bearing the new glucosylated multivalent conjugate, as confirmed by ELISA. These results may offer a promising proof-of-concept for the selective purification of high affinity autoantibodies from sera of autoimmune patients, in general, and of specific high affinity antibodies against a minimally glcosylated epitope Asn(Glc) from sera of multiple sclerosis (MS) patients, in particular., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Immune response to biologics treatment, while widely reported, yet fails to correlate with clinical outcomes and assay to assay comparison is often not possible. Hence, we developed a new peptide based-detection assay to stratify pediatric patients with juvenile idiopathic arthritis (JIA) or chronic non-infectious uveitis (CNU) and monitor anti-drug antibodies (ADAbs) formed as part of an immune response to treatment with the fully human monoclonal therapeutic antibody Adalimumab. Adalimumab derived synthetic peptides were optimized for maximum immunogenicity and were tested by SP-ELISA on a development cohort of 18 JIA and CNU treated patients. The two best performing peptides able to differentiate patient groups were selected for evaluation with a larger scale ELISA testing on a total of 29 sera from pediatric patients with JIA or CNU. The results of this peptide-based assay were compared to an in-house developed SPR biosensor ADAbs assay and a commercially available bridging ELISA. The first peptide, termed HC3, was able to positively detect ADAbs in 7 out of the 29 sera, while the second peptide, called LC3, was able to detect ADAbs in 11 out of 29 sera in the evaluation group. Following statistical data evaluation, it has been found that the detection of ADAbs using the peptide-based ELISA assay positively correlates with disease progression and remission. Two synthetic peptides derived from Adalimumab may provide a beneficial tool to clinicians for monitoring patient response to such treatment and taking informed decisions for treatment alternatives., (© 2021. The Author(s).)

Peptides mimicking antigenic epitopes targeted by antibodies can be powerful tools to be used as antigen surrogates for the specific diagnosis and treatment of autoimmune diseases. Obtaining structural insights about the nature of peptide-antibody interaction in complex mixtures such as sera is a critical goal. In multiple sclerosis (MS), we previously demonstrated that the N-linked β-d-glucopyranosyl moieties (N-Glc) containing epitopes in nontypeable Haemophilus influenzae adhesin C-terminal portion HMW1(1205-1526) were essential for high-affinity antibody binding in a subpopulation of MS patients. With the aim of developing peptide probes and assessing their binding properties to antibodies from sera of representative patients, we performed the systematic analysis of synthetic peptides based on HMW1(1347-1354) fragment bearing one or two N-Glc respectively on Asn-1349 and/or Asn-1352. The N-glucosylated nonapeptides efficiently bind to IgG antibodies, displaying IC 50 in the range 10 -8 -10 -10 M by competitive indirect enzyme-linked immunosorbent assay (ELISA) in three representative MS patient sera. We selected the di-N-glucosylated adhesin peptide Ac-KAN (Glc)VTLN (Glc)TT-NH 2 as the shortest sequence able to inhibit high-avidity interaction with N-Glc targeting IgM antibodies. Nuclear magnetic resonance (NMR)- and circular dichroism (CD)-based characterization showed that the binding properties of these antigens could not be ascribed to structural differences induced by the presence of up to two N-glucosyl moieties. Therefore, the antibody binding is not easily correlated to the position of the sugar or to a determined conformation in water., (© 2020 European Peptide Society and John Wiley & Sons, Ltd.)

We report herein a novel ChemMatrix ® Rink resin functionalised with two phenylboronate (PhB) moieties linked on the N -α and N -ε amino functions of a lysine residue to specifically capture deoxyfructosylated peptides, compared to differently glycosylated peptides in complex mixtures. The new PhB-Lys(PhB)-ChemMatrix ® Rink resin allows for exploitation of the previously demonstrated ability of cis diols to form phenylboronic esters. The optimised capturing and cleavage procedure from the novel functionalised resin showed that only the peptides containing deoxyfructosyl-lysine moieties can be efficiently and specifically detected by HR-MS and MS/MS experiments. We also investigated the high-selective affinity to deoxyfructosylated peptides in an ad hoc mixture containing unique synthetic non-modified peptides and in the hydrolysates of human and bovine serum albumin as complex peptide mixtures. We demonstrated that the deoxyfructopyranosyl moiety on lysine residues is crucial in the capturing reaction. Therefore, the novel specifically-designed PhB-Lys(PhB)-ChemMatrix ® Rink resin, which has the highest affinity to deoxyfructosylated peptides, is a candidate to quantitatively separate early glycation peptides from complex mixtures to investigate their role in diabetes complications in the clinics.

Background: Mitochondria play a role in type 1 diabetes (T1D) particularly in the treatment and prevention of disorder consequences. Due to their demonstrated role in diabetes pathology, mitochondrial proteins can be an interesting starting point to study candidate antigens in T1D. We investigated the role of relevant post-translational modifications (PTM) on a synthetic mitochondrial peptide as putative antigen., Methods: The antibody response in T1D was evaluated by solid phase-ELISA using a collection of synthetic peptides bearing different PTMs. We investigated the role of lipoylation, phosphorylation, and glycosylation. The PTMs were introduced at position 173 of the mitochondrial pyruvate dehydrogenase E2 complex peptide PDC-E2(167-184) and at position 7 of a structure-based designed β-turn peptide as an irrelevant sequence to investigate the role of the specific PDC-E2 peptide sequence., Results: IgM titres in 31 T1D patients were higher than IgGs to all the synthetic PTM peptides. Results demonstrated the crucial role of lysine lipoamide, serine O-phosphorylation, and O-glycosylation into the PDC-E2(167-184) peptide sequence for IgM antibody recognition., Conclusions: Results highlight the importance of immune dysregulation in T1D, furthermore, if confirmed in a large number of patients, they will contribute to add novel diagnostic markers for the understanding the physiopathology of the disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In autoimmune diseases, there have been proposals that exogenous “molecular triggers”, i.e., specific ‘non-self antigens’ accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies. The etiology of multiple sclerosis (MS) remains unclear. However, epidemiologic data suggest that exposure to infectious agents may be associated with increased MS risk and progression may be linked to exogenous, bacterially-derived, antigenic molecules, mimicking mammalian cell surface glycoconjugates triggering autoimmune responses. Previously, antibodies specific to a gluco-asparagine (N-Glc) glycopeptide, CSF114(N-Glc), were identified in sera of an MS patient subpopulation. Since the human glycoproteome repertoire lacks this uniquely modified amino acid, we turned our attention to bacteria, i.e., Haemophilus influenzae, expressing cell-surface adhesins including N-Glc, to establish a connection between H. influenzae infection and MS. We exploited the biosynthetic machinery from the opportunistic pathogen H. influenzae (and the homologous enzymes from A. pleuropneumoniae) to produce a unique set of defined glucosylated adhesin proteins. Interestingly we revealed that a hyperglucosylated protein domain, based on the cell-surface adhesin HMW1A, is preferentially recognized by antibodies from sera of an MS patient subpopulation. In conclusion the hyperglucosylated adhesin is the first example of an N-glucosylated native antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS.

The interaction of lipid environments with the type I' β-turn peptide structure called CSF114 and its N-glucosylated form CSF114(Glc), previously developed as a synthetic antigenic probe recognizing specific autoantibodies in a subpopulation of multiple sclerosis patients' serum, was investigated by fluorescence spectroscopy and electrochemical experiments using large unilamellar vesicles, mercury supported lipid self-assembled monolayers (SAMs) and tethered bilayer lipid membranes (tBLMs). The synthetic antigenic probe N-glucosylated peptide CSF114(Glc) and its unglucosylated form interact with the polar heads of lipid SAMs of dioleoylphosphatidylcholine at nonzero transmembrane potentials, probably establishing a dual electrostatic interaction of the trimethylammonium  and phosphate groups of the phosphatidylcholine polar head with the Glu⁵ and His⁸ residues on the opposite ends of the CSF114(Glc) β-turn encompassing residues 6-9. His⁸ protonation at pH 7 eliminates this dual interaction. CSF114(Glc) is adsorbed on top of SAMs of mixtures of dioleoylphosphatidylcholine with sphingomyelin, an important component of myelin, whose proteins are hypothesized to undergo an aberrant N-glucosylation triggering the autoimmune response. Incorporation of the type I' β-turn peptide structure CSF114 into lipid SAMs by potential scans of electrochemical impedance spectroscopy induces defects causing a slight permeabilization toward cadmium ions. The N-glucopeptide CSF114(Glc) does not affect  tBLMs to a detectable extent.

Primary biliary cirrhosis is an immune-mediated chronic liver disease whose diagnosis relies on the detection of serum antimitochondrial antibodies directed against a complex set of proteins, among which pyruvate dehydrogenase complex is considered the main autoantigen. We studied the immunological role of the lipoyl domain of this protein using synthetic lipoylated peptides, showing that the lipoyl chain chirality does not affect autoantibody recognition and, most importantly, confirming that both lipoylated and unlipoylated peptides are able to recognize specific autoantibodies in patients sera. In fact, 74% of patients sera recognize at least one of the tested peptides but very few positive sera recognized exclusively the lipoylated peptide, suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope. These results are supported by a conformational analysis showing that the lipoyl moiety of pyruvate dehydrogenase complex appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex antigenic epitope. A preliminary analysis of the specificity of the two most active peptides indicates that they could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex positivity pattern detected in immunofluorescence.

Rett syndrome (RTT), a neurodevelopmental disorder affecting exclusively (99%) female infants, is associated with loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2) and, more rarely, cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1). In this study, we aimed to evaluate the function of the immune system by measuring serum immunoglobulins (IgG and IgM) in RTT patients (n = 53) and, by comparison, in age-matched children affected by non-RTT pervasive developmental disorders (non-RTT PDD) (n = 82) and healthy age-matched controls (n = 29). To determine immunoglobulins we used both a conventional agglutination assay and a novel ELISA based on antibody recognition by a surrogate antigen probe, CSF114(Glc), a synthetic N-glucosylated peptide. Both assays provided evidence for an increase in IgM titer, but not in IgG, in RTT patients relative to both healthy controls and non-RTT PDD patients. The significant difference in IgM titers between RTT patients and healthy subjects in the CSF114(Glc) assay (P = 0.001) suggests that this procedure specifically detects a fraction of IgM antibodies likely to be relevant for the RTT disease. These findings offer a new insight into the mechanism underlying the Rett disease as they unveil the possible involvement of the immune system in this pathology.

Sophisticated approaches have recently led to the identification of novel autoantigens associated with Multiple Sclerosis (MuS), e.g. neurofascin, contactin, CNPase, and other T-cell receptor membrane anchored proteins. These putative antigens, although differing from the conventional myelin derivatives, are conceptually based on an animal model of experimental autoimmune encephalomyelitis. In this report we describe the identification of putative antigens based on their recognition by autoantibodies isolated from MuS patient serum. In a previous work from this laboratory we have shown that a peptide probe, named CSF114(Glc), specifically identifies serum autoantibodies in a subset of MuS patients, representing ∼30% of the patient population. The autoantibodies, purified from MuS patients' sera (six), through CSF114(Glc) affinity chromatography, detected three immunoreactive protein bands present in the rat brain. Proteomic analysis of the immunoreactive bands, involving MALDI and MS/MS techniques, revealed the presence of four proteins distinguishable by their mass: alpha fodrin, alpha actinin 1, creatine kinase, and CNPase. The immunoreactive profile of these rat brain proteins was compared with that of commercially available standard proteins by challenging against either CSF114(Glc) purified MuS autoantibodies, or monoclonal antibodies. Further discrimination among the rat brain proteins was provided by the following procedure: whereas monoclonal antibodies recognized all rat brain proteins, isolated MuS specific antibodies recognize only alpha actinin 1 as a putative antigen. In fact, alpha actinin 1 displayed a robust immunoreactive response against all MuS patients' sera examined, whereas the other three bands were not consistently detectable. Thus, alpha actinin 1, a cytoskeleton protein implicated in inflammatory/degenerative autoimmune diseases (lupus nephritis and autoimmune hepatitis) might be regarded as a novel MuS autoantigen, perhaps a prototypic biomarker for the inflammatory/degenerative process typical of the disease.